(1/145) Manganese sulfate-dependent glycosylation of endogenous glycoproteins in human skeletal muscle is catalyzed by a nonglucose 6-P-dependent glycogen synthase and not glycogenin.

Glycogenin, a Mn2+-dependent, self-glucosylating protein, is considered to catalyze the initial glucosyl transfer steps in glycogen biogenesis. To study the physiologic significance of this enzyme, measurements of glycogenin mediated glucose transfer to endogenous trichloroacetic acid precipitable material (protein-bound glycogen, i.e., glycoproteins) in human skeletal muscle were attempted. Although glycogenin protein was detected in muscle extracts, activity was not, even after exercise that resulted in marked glycogen depletion. Instead, a MnSO4-dependent glucose transfer to glycoproteins, inhibited by glycogen and UDP-pyridoxal (which do not affect glycogenin), and unaffected by CDP (a potent inhibitor of glycogenin), was consistently detected. MnSO4-dependent activity increased in concert with glycogen synthase fractional activity after prolonged exercise, and the MnSO4-dependent enzyme stimulated glucosylation of glycoproteins with molecular masses lower than those glucosylated by glucose 6-P-dependent glycogen synthase. Addition of purified glucose 6-P-dependent glycogen synthase to the muscle extract did not affect MnSO4-dependent glucose transfer, whereas glycogen synthase antibody completely abolished MnSO4-dependent activity. It is concluded that: (1) MnSO4-dependent glucose transfer to glycoproteins is catalyzed by a nonglucose 6-P-dependent form of glycogen synthase; (2) MnSO4-dependent glycogen synthase has a greater affinity for low molecular mass glycoproteins and may thus play a more important role than glucose 6-P-dependent glycogen synthase in the initial stages of glycogen biogenesis; and (3) glycogenin is generally inactive in human muscle in vivo.  (+info)

(2/145) Modes of action of acarbose hydrolysis and transglycosylation catalyzed by a thermostable maltogenic amylase, the gene for which was cloned from a Thermus strain.

A maltogenic amylase gene was cloned in Escherichia coli from a gram-negative thermophilic bacterium, Thermus strain IM6501. The gene encoded an enzyme (ThMA) with a molecular mass of 68 kDa which was expressed by the expression vector p6xHis119. The optimal temperature of ThMA was 60 degrees C, which was higher than those of other maltogenic amylases reported so far. Thermal inactivation kinetic analysis of ThMA indicated that it was stabilized in the presence of 10 mM EDTA. ThMA harbored both hydrolysis and transglycosylation activities. It hydrolyzed beta-cyclodextrin and starch mainly to maltose and pullulan to panose. ThMA not only hydrolyzed acarbose, an amylase inhibitor, to glucose and pseudotrisaccharide (PTS) but also transferred PTS to 17 sugar acceptors, including glucose, fructose, maltose, cellobiose, etc. Structural analysis of acarbose transfer products by using methylation, thin-layer chromatography, high-performance ion chromatography, and nuclear magnetic resonance indicated that PTS was transferred primarily to the C-6 of the acceptors and at lower degrees to the C-3 and/or C-4. The transglycosylation of sugar to methyl-alpha-D-glucopyranoside by forming an alpha-(1,3)-glycosidic linkage was demonstrated for the first time by using acarbose and ThMA. Kinetic analysis of the acarbose transfer products showed that the C-4 transfer product formed most rapidly but readily hydrolyzed, while the C-6 transfer product was stable and accumulated in the reaction mixture as the main product.  (+info)

(3/145) The AcbC protein from Actinoplanes species is a C7-cyclitol synthase related to 3-dehydroquinate synthases and is involved in the biosynthesis of the alpha-glucosidase inhibitor acarbose.

The putative biosynthetic gene cluster for the alpha-glucosidase inhibitor acarbose was identified in the producer Actinoplanes sp. 50/110 by cloning a DNA segment containing the conserved gene for dTDP-D-glucose 4,6-dehydratase, acbB. The two flanking genes were acbA (dTDP-D-glucose synthase) and acbC, encoding a protein with significant similarity to 3-dehydroquinate synthases (AroB proteins). The acbC gene was overexpressed heterologously in Streptomyces lividans 66, and the product was shown to be a C7-cyclitol synthase using sedo-heptulose 7-phosphate, but not ido-heptulose 7-phosphate, as its substrate. The cyclization product, 2-epi-5-epi-valiolone ((2S,3S,4S,5R)-5-(hydroxymethyl)cyclohexanon-2,3,4,5-tetrol), is a precursor of the valienamine moiety of acarbose. A possible five-step reaction mechanism is proposed for the cyclization reaction catalyzed by AcbC based on the recent analysis of the three-dimensional structure of a eukaryotic 3-dehydroquinate synthase domain (Carpenter, E. P., Hawkins, A. R., Frost, J. W., and Brown, K. A. (1998) Nature 394, 299-302).  (+info)

(4/145) Acarbose, a pseudooligosaccharide, is transported but not metabolized by the maltose-maltodextrin system of Escherichia coli.

The pseudooligosaccharide acarbose is a potent inhibitor of amylases, glucosidases, and cyclodextrin glycosyltransferase and is clinically used for the treatment of so-called type II or insulin-independent diabetes. The compound consists of an unsaturated aminocyclitol, a deoxyhexose, and a maltose. The unsaturated aminocyclitol moiety (also called valienamine) is primarily responsible for the inhibition of glucosidases. Due to its structural similarity to maltotetraose, we have investigated whether acarbose is recognized as a substrate by the maltose/maltodextrin system of Escherichia coli. Acarbose at millimolar concentrations specifically affected the growth of E. coli K-12 on maltose as the sole source of carbon and energy. Uptake of radiolabeled maltose was competitively inhibited by acarbose, with a Ki of 1.1 microM. Maltose-grown cells transported radiolabeled acarbose, indicating that the compound is recognized as a substrate. Studying the interaction of acarbose with purified maltoporin in black lipid membranes revealed that the kinetics of acarbose binding to LamB is asymmetric. The on-rate of acarbose is approximately 30 times lower when the molecule enters the pore from the extracellular side than when it enters from the periplasmic side. Acarbose could not be utilized as a carbon source since the compound alone was not a substrate of amylomaltase (MalQ) and was only poorly attacked by maltodextrin glucosidase (MalZ).  (+info)

(5/145) A randomized double-blind trial of acarbose in type 2 diabetes shows improved glycemic control over 3 years (U.K. Prospective Diabetes Study 44)

OBJECTIVE: To determine the degree to which alpha-glucosidase inhibitors, with their unique mode of action primarily reducing postprandial hyperglycemia, offer an additional therapeutic approach in the long-term treatment of type 2 diabetes. RESEARCH DESIGN AND METHODS: We studied 1,946 patients (63% men) who were previously enrolled in the U.K. Prospective Diabetes Study (UKPDS). The patients were randomized to acarbose (n = 973), titrating to a maximum dose of 100 mg three times per day, or to matching placebo (n = 973). Mean +/- SD age was 59 +/- 9 years, body weight 84 +/- 17 kg, diabetes duration 7.6 +/- 2.9 years, median (interquartile range) HbA1c 7.9% (6.7-9.5), and fasting plasma glucose (FPG) 8.7 mmol/l (6.8-11.1). Fourteen percent of patients were treated with diet alone, 52% with monotherapy, and 34% with combined therapy. Patients were monitored in UKPDS clinics every 4 months for 3 years. The main outcome measures were HbA1c, FPG, body weight, compliance with study medication, incidence of side effects, and frequency of major clinical events. RESULTS: At 3 years, a lower proportion of patients were taking acarbose compared with placebo (39 vs. 58%, P < 0.0001), the main reasons for noncompliance being flatulence (30 vs. 12%, P < 0.0001) and diarrhea (16 vs. 8%, P < 0.05). Analysis by intention to treat showed that patients allocated to acarbose, compared with placebo, had 0.2% significantly lower median HbA1c at 3 years (P < 0.001). In patients remaining on their allocated therapy, the HbA1c difference at 3 years (309 acarbose, 470 placebo) was 0.5% lower median HbA1c (8.1 vs. 8.6%, P < 0.0001). Acarbose appeared to be equally efficacious when given in addition to diet alone; in addition to monotherapy with a sulfonylurea, metformin, or insulin; or in combination with more complex treatment regimens. No significant differences were seen in FPG, body weight, incidence of hypoglycemia, or frequency of major clinical events. CONCLUSIONS: Acarbose significantly improved glycemic control over 3 years in patients with established type 2 diabetes, irrespective of concomitant therapy for diabetes. Careful titration of acarbose is needed in view of the increased noncompliance rate seen secondary to the known side effects.  (+info)

(6/145) Changes of fermentation pathways of fecal microbial communities associated with a drug treatment that increases dietary starch in the human colon.

Acarbose inhibits starch digestion in the human small intestine. This increases the amount of starch available for microbial fermentation to acetate, propionate, and butyrate in the colon. Relatively large amounts of butyrate are produced from starch by colonic microbes. Colonic epithelial cells use butyrate as an energy source, and butyrate causes the differentiation of colon cancer cells. In this study we investigated whether colonic fermentation pathways changed during treatment with acarbose. We examined fermentations by fecal suspensions obtained from subjects who participated in an acarbose-placebo crossover trial. After incubation with [1-13C]glucose and 12CO2 or with unlabeled glucose and 13CO2, the distribution of 13C in product C atoms was determined by nuclear magnetic resonance spectrometry and gas chromatography-mass spectrometry. Regardless of the treatment, acetate, propionate, and butyrate were produced from pyruvate formed by the Embden-Meyerhof-Parnas pathway. Considerable amounts of acetate were also formed by the reduction of CO2. Butyrate formation from glucose increased and propionate formation decreased with acarbose treatment. Concomitantly, the amounts of CO2 reduced to acetate were 30% of the total acetate in untreated subjects and 17% of the total acetate in the treated subjects. The acetate, propionate, and butyrate concentrations were 57, 20, and 23% of the total final concentrations, respectively, for the untreated subjects and 57, 13, and 30% of the total final concentrations, respectively, for the treated subjects.  (+info)

(7/145) Structure-function relationships in glucoamylases encoded by variant Saccharomycopsis fibuligera genes.

The mutation Gly467-->Ser in Glu glucoamylase was designed to investigate differences between two highly homologous wild-type Saccharomycopsis fibuligera Gla and Glu glucoamylases. Gly467, localized in the conserved active site region, S5, is replaced by Ser in the Gla glucoamylase. These amino acid residues are the only two known to occupy this position in the elucidated glucoamylase sequences. The data from the kinetic analysis revealed that replacement of Gly467 with Ser in Glu glucoamylase decreased the kcat towards all substrates tested to values comparable with those of the Gla enzyme. Moreover, the mutant glucoamylase appeared to be less stable compared to the wild-type Glu glucoamylase with respect to thermal unfolding. Microcalorimetric titration studies of the interaction with the inhibitor acarbose indicated differences in the binding between Gla and Glu enzymes. The Gla glucoamylase, although less active, binds acarbose stronger (Ka congruent with 10(13).M(-1)) than the Glu enzyme (Ka congruent with 10(12).M(-1)). In all enzymes studied, the binding of acarbose was clearly driven by enthalpy, with a slightly favorable entropic contribution. The binding of another glucoamylase inhibitor, 1-deoxynojirimycin, was about 8-9 orders of magnitude weaker (Ka congruent with 10(4).M(-1)) than that of acarbose. From comparison of kinetic parameters for the nonglycosylated and glycosylated enzymes it can be deduced that the glycosylation does not play a critical role in enzymatic activity. However, results from differential scanning calorimetry demonstrate an important role of the carbohydrate moiety in the thermal stability of glucoamylase.  (+info)

(8/145) Mechanism of porcine pancreatic alpha-amylase inhibition of amylose and maltopentaose hydrolysis by kidney bean (Phaseolus vulgaris) inhibitor and comparison with that by acarbose.

The effects of Phaseolus vulgaris inhibitor (alpha-AI) on the amylose and maltopentaose hydrolysis catalysed by porcine pancreatic alpha-amylase (PPA) were investigated. Based on a statistical analysis of the kinetic data and using the general velocity equation, which is valid at equilibrium for all types of inhibition in a single-substrate reaction, it was concluded that the inhibitory mode is of the mixed noncompetitive type involving two molecules of inhibitor. In line with this conclusion, the Lineweaver-Burk primary plots intersect in the second quadrant and the secondary plots of the slopes and the intercepts versus the inhibitor concentrations are parabolic curves, whether the substrate used was amylose or maltopentaose. A specific inhibition model of the mixed noncompetitive type applies here. This model differs from those previously proposed for acarbose [Al Kazaz, M., Desseaux, V., Marchis-Mouren, G., Payan, F., Forest, E. & Santimone, M. (1996) Eur. J. Biochem. 241, 787-796 and Al Kazaz, M., Desseaux, V., Marchis-Mouren, G., Prodanov, E. & Santimone, M. (1998) Eur. J. Biochem. 252, 100-107]. In particular, with alpha-AI, the inhibition takes place only when PPA and alpha-AI are preincubated together before the substrate is added. This shows that the inhibitory PPA-alphaAI complex is formed during the preincubation period. Secondly, other inhibitory complexes are formed, in which two molecules of inhibitor are bound to either the free enzyme or the enzyme-substrate complex. The catalytic efficiency was determined both with and without inhibitor. Using the same molar concentration of inhibitor, alpha-AI was found to be a much stronger inhibitor than acarbose. However, when the inhibitor amount is expressed on a weight basis (mg x L-1), the opposite conclusion is drawn. In addition, limited proteolysis was performed on PPA alone and on the alpha-AI-PPA complex. The results show that, in the complex, PPA is more sensitive to subtilisin attack, and shorter fragments are obtained. These data reflect the conformational changes undergone by PPA as the result of the protein inhibitor binding, which differ from those previously observed with acarbose.  (+info)

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